Field of the Invention
The present invention relates to a method and a test kit for detection of specific nucleic acid sequences with the steps of amplification, hybridization by means of probes, and detection of the hybridization event; wherein the detection of the hybridization event takes place on a solid phase outside the reaction vessel for amplification/hybridization.
Description of the Related Art
Genetic diagnostics has become an indispensable tool of modern medical laboratory diagnostics, forensic diagnostics, veterinary medical laboratory diagnostics or food and environmental diagnostics.
Genetic diagnostics was revolutionized with the invention of PCR technology, with which it is possible to amplify any arbitrary nucleic acid sequence specifically.
The use of PCR covers a diversity of methods, which in combination with the PCR technology additionally permit specific detection of completed amplification. Especially the requirements of an exact genetic diagnosis must make use of methods that ensure that a generated amplification product also corresponds to the target sequence that is specifically to be detected. The widespread use of visualization of a PCR product by means of gel electrophoresis is not sufficient for this purpose.
One possibility for detection of specific nucleic acid sequences in a way that in principle can be achieved very rapidly and without great experimental time and effort is what are known as real-time PCR methods. In this case the amplification reaction is directly coupled with the actual detection reaction.
A widely used method for detection of specific nucleic acids is light cycler technology (Roche). For this purpose Roche has developed special hybridization probes, consisting of two different oligonucleotides, each labeled with only one fluorochrome. The acceptor is located at the 3′-end of the one probe and the other oligonucleotide has a donor at the 5′-end. The probes are chosen such that they both bind to the same DNA strand, the distance between acceptor and donor being permitted to be at most 1 to 5 nucleotides, so that what is known as the FRET effect can occur. The fluorescence is measured during the annealing step, in which only light of this wavelength is detectable as long as both probes are bound to the DNA. In this system the melting point of both probes should be identical. Because of the use of two hybridizing probes in addition to the primers used, the specificity of this detection system is extremely high.
A further real-time PCR application for detection of specific nucleic acid targets can be performed with what are known as double-dye probes, which are disclosed in U.S. Pat. Nos. 5,210,015 and 5,487,972 (TaqMan probes), both of which are incorporated by reference. Double-dye probes carry two fluorochromes on one probe. The reporter dye is located in this case at the 5′-end and the quencher dye at the 3′-end. In addition, a phosphate group is also located at the 3′-end of the probe if necessary, so that the probe cannot function as a primer during elongation. As long as the probe is intact, the released light intensity is low, since almost the entire light energy produced after excitation of the reporter is absorbed and transformed by virtue of the spatial proximity of the quencher. The emitted light of the reporter dye is “quenched”, or in other words extinguished. This FRET effect is preserved even after the probe has bonded to the complementary DNA strand. During the elongation phase, the polymerase encounters the probe and hydrolyzes it. The ability of the polymerase to hydrolyze an oligonucleotide (or a probe) during strand synthesis is known as 5′-3′ exonuclease activity. Not all polymerases have 5′-3′ exonuclease activity (Taq and Tth polymerase). This principle was first described for the Taq polymerase. The principle is known as the TaqMan principle. After probe hydrolysis, the reporter dye is no longer located in spatial proximity to the quencher. The emitted fluorescence is now no longer transformed and this fluorescence increase is measured.
A further option for specific detection of amplification products by means of real-time PCR technology consists in the use of intercalating dyes (ethidium bromide, Hoechst 33258, Yo-Pro-1 or SYBR Green™ and the like). After being excited by high-energy UV light, these dyes emit light in the lower-energy visible wavelength region (fluorescence). If the dye is present as a free dye in the reaction mixture, the emission is very weak. Only by intercalation of the dye, whereby it fits into the small furrows of double-strand DNA molecules, is the light emission greatly intensified. The dyes are inexpensive and universally usable, since in principle any PCR reaction can be followed in real time with them. In addition, they have high signal strength, since every DNA molecule binds several dye molecules. From the advantages, however, there also results an extreme disadvantage for application: In principle it is not possible by means of intercalating dyes to distinguish between correct product and amplification artifacts (such as primer dimers or defective products). While primer dimers and other artifacts are being formed, they naturally also bind intercalating dyes and thus lead to an unspecific increase in fluorescence even in negative samples. However, a clear differentiation between specific amplification event or artifact is absolutely necessary. In order to achieve this in any case, what is known as a melting-point analysis is performed at the end of the actual PCR reaction. For this purpose the reaction mixture is heated in steps of 1 degree from 50° C. to 90° C. The fluorescence is measured continuously during this process. The point at which double-strand DNA melts is characterized by a decrease (peak) of the fluorescence of the intercalating dye, since the intercalating dye dissociates from the single-strand DNA. When the PCR is optimally adjusted, a melting-point peak that tapers sharply is to be expected. This melting point represents the specific product to be expected. Products of different sizes and products of other sequences have different melting points.
When the fluorescence is plotted graphically against temperature, the fluorescence decrease of the two products can be perceived as two separate melting points. Thus this system gains specificity and makes it possible to distinguish a specific amplification product from artifacts. In this way it is possible to distinguish even between homozygotes (single peak) and heterozygotes (two peaks).
Furthermore, it is also possible to achieve quantitation of the targets to be detected by means of REAL-time PCR applications.
As already explained, the described methods fulfill the need for specific detection of an amplification product.
A great disadvantage, however, is that they are implemented on very expensive instrumental platforms, which have to unite the process of amplification and that of subsequent optical detection, in a manner corresponding to the problem, in one hardware solution. Furthermore, many of these described detection methods are still based on real-time tracking of the amplification process. On the basis of this strategy, even workup of the measured fluorescence values takes place in the course of the amplification reaction. It is clear to those skilled in the art that, in this connection, an enormously large body of analysis algorithms must also be integrated into real-time systems. Ultimately this explains the high financial expenditure that must be invested for the use of real-time PCR systems, Also ultimately, the operation of such instrumental systems requires a high degree of expertise.
Besides the described diagnostic detections based on REAL-time PCR, however, alternative variants for specific detection of nucleic acids also exist.
An example of less expensive methods for detection of nucleic acids in this connection is PCR-ELISA. In this method, the DNA sequence to be examined is amplified and the generated DNA fragment is then covalently immobilized on a solid phase (such as microtiter plates or strips), denatured to a single strand and hybridized with a sequence-specific probe. Successful binding of the probe can be visualized with an antibody-mediated color reaction. Another variant is based on immobilizing the probes on a solid phase, denaturing the PCR product and then bringing it into contact with the immobilized probe. Detection of a completed hybridization event takes place by analogy with the first variant of the method.
In principle, PCR-ELISA methods are easy to perform, but they comprise multiple procedural steps. Besides the time needed to perform the PCR, therefore, several hours of working time are also needed to perform the subsequent detection method. Such a method usually needs 8 hours and therefore is also not suitable as a rapid test.
Furthermore, some items of equipment are also needed, such as a temperature-control station, what is known as a washer, or even a measuring instrument for detection of the hybridization signal. Furthermore, other special instruments or special consumable materials may also be necessary.
Further simple methods for detection of amplification products are based on amplification of the target sequences and subsequent hybridization of amplification products on a membrane. These methods also have several variants known to those skilled in the art. Once again, however, these methods are also laborious to perform, need a large number of procedural steps to be performed and therefore are not suitable as rapid tests. This then also applies to the use of biochip strategies, which use hybridization of PCR products with hybridization probes for detection of the specificity. These methods also are laborious and associated with very expensive instrumental platforms.
A distinct reduction of working steps is disclosed in Korean Patent 1020060099022 A (Method and kit for rapid and accurate detection and analysis of nucleotide sequence with naked eye by using membrane lateral flow analysis).
In this case what is known as a lateral flow method is used to detect nucleic acids. This method also makes use of the technology of hybridization of nucleic acids on a solid phase. Advantageously, a lateral flow method has a small, handy test format (strip test).
In contrast to the above patent specification, a very fast detection method, which also makes use of detection of amplification products by means of a test strip and is commercially available, is in turn based on a completely different principle. In this case the PCR reaction is performed with a biotinylated primer and a non-biotinylated primer. After the PCR has been performed, there is obtained a PCR product that is therefore biotin-labeled at one end. Detection is achieved using a test strip (for example of the Millenia Co.), which contains two separate binding sites: a streptavidin site for coupling the biotin-labeled DNA strand and an FITC binding site for functional control of the test strip.
Detection of the PCR product is achieved by denaturing the PCR mixture on completion of the PCR and hybridizing it with a probe complementary to the biotin-labeled DNA strand. The probe is FITC-labeled.
For detection, the PCR mixture is mixed with a running buffer and applied on the test strip. According to the description of the test, the biotinylated DNA strand binds to the streptavidin binding site of the strip. Detection takes place via the FITC labeling of the probe hybridized with the DNA strand. A typical signal in the form of a strip is developed. This signal is supposed to be the specific detection of the amplification product. However, the method does not combine hybridization of the probe with the PCR process but instead performs the latter process as a separate procedural step. However, the method suffers from a fundamental and dramatic error source.
Detection of the target nucleic acid to be detected is not specific. The reason is that artifacts such as primer dimers are formed during PCR and naturally also bind specifically to the streptavidin binding sites of the test strip, and so they can cause a positive reaction just as does a specific PCR product.
International Document WO 2004/092342 A2 describes the technology of the lateral-flow assay, which is incorporated by reference. As examples of application to molecular biology, there are used already known and in some cases commercially available technologies, which are adapted to the lateral-flow assay of that invention. In Example 1 of WO 2004/092342 A2, one of the RT reactions and subsequent amplification is performed with two labeled primers. This method may lead to false-positive results due to primer-dimer formation and mispriming. The second option (FIG. 20d-e) represents a subsequent hybridization with two labeled probes. The problem of primer-dimer formation and mispriming is not acknowledged in that publication.
The important problem of false-positive results due to primer-dimer formation was correctly recognized in the publication of Kozwich, et al. (Development of a novel, rapid integrated Cryptosporidium parvum detection assay. Appl. Environ. Microbiol. (2000) 66 (7) 2711 7, page 2712, right column, 2nd par., FIG. 3), incorporated by reference. The solution of the problem (nested PCR with the labeled and non-labeled primers) differs in principle from the solution of the present invention, for which protection is applied for herewith (one labeled primer and one labeled probe). The solution proposed in the publication of Kozwich, et al. excludes the formation of primer dimers only as a matter of probability but not of principle. The mispriming that occurs so often is also not completely excluded as an error source in the solution proposed by Kozwich, et al.
All of the described alternative methods for detection of nucleic acid sequences without REAL-time PCR technologies therefore also have a substantial common feature, regardless of the considerable manual working effort that is still necessary. The necessary hybridization reaction between PCR product and specific probe always takes place outside the PCR process. This feature is at the base of all of these methods. A major advantage of REAL-time PCR technologies, however, is precisely that the processes of amplification and specific hybridization take place in one reaction vessel, and so the processes of amplification and hybridization are not disconnected. Furthermore, amplification artifacts frequently lead to a false-positive signal in these cases.